The invention is in the fields of vaccine development and application and relates to attenuated live vaccine vectors, more specifically to such vectors based on or derived from genetically modified influenza A virus strains, and to the manufacture of recominant influenza viruses and vaccines.
Influenza viruses are segmented negative-strand RNA viruses and belong to the Orthomyxoviridae family. Influenza A virus consists of 9 structural proteins and codes additionally for one nonstructural NS1 protein with regulatory functions. The non-structural NS1 protein is synthesized in large quantities during the reproduction cycle and is localized in the cytosol and nucleus of the infected cells. The segmented nature of the viral genome allows the mechanism of genetic reassortment (exchange of genome segments) to take place during mixed infection of a cell with different viral strains. Several features make influenza viruses attractive candidates for the development of effective live vaccine vectors against different diseases:
(i) influenza viruses induce strong cellular and humoral immune responses, at the systemic and the mucosal level against viral proteins following infection;
(ii) influenza virus as an RNA virus does not contain a DNA phase in its replication cycle. Therefore chromosomal integration of viral genes into the host can be excluded;
(iii) many different influenza virus subtypes are available. Since antibodies against these different subtypes show no or little crossreactivity, pre-existing immunity to the viral vector in the host, which is frequently a problem for other live vectors, can be circumvented. Also, effective booster immunizations with different subtype influenza viruses expressing the same antigens might be possible; and
(iv) attenuated influenza viruses as live influenza vaccines, which were shown to be safe and immunogenic in humans, are available.
Until now the main problem of utilizing influenza virus as a vector concerns the size of the virus genome and its limited capacity to tolerate foreign sequences. Among ten influenza viral proteins, only the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) have been successfully engineered for stable expression of foreign epitopes. Since influenza virus tolerates an insertion of only approximately 10 amino acids into its HA molecule, there is only a limited possibility to influence the conformation properties of inserted epitopes which would probably be better presented if longer sequences would be introduced. Besides, surface influenza glycoproteins such as HA or NA cannot be considered as optimal targets for the presentation of foreign sequences because of their association with antigenic properties of the viruses. An HA live virus construct containing a desired foreign antigen is not applicable for boosting immunizations (e.g., by second and further administrations) because of the pre-existing immunity against the HA caused by the first immunization or by a natural virus infection. A booster immunization would be possible only upon introduction of the desired antigenic structure into another HA molecule belonging to a different influenza virus subtype. It is evident that such a process is difficult, laborious and extremely time consuming and therefore unlikely to be suitable for routine vaccine preparation
Preceding investigations in connection with the present invention have indicated that the NS gene of influenza A virus may be a promising alternative to HA as a viral carrier for presenting a desired foreign antigen to the animal or human immune system. The recently established method of reverse genetics (Egorov et al., 1998, J Virol 72(8), 6437-41) allows to rescue influenza viruses containing long deletions or insertions of foreign sequences at the carboxyl side of the non-structural Protein 1 (NS1 protein). NS1 protein is abundant in influenza virus-infected cells and stimulates cytotoxic T-lymphocyte (CTL) responses as well as antibody responses during the natural course of influenza virus infection.
Further details about the influenza virus NS gene can be found in WO 99/64571. Additionally, WO 99/64571 discloses that attenuated influenza A virus transfectants containing knockout deletions of the entire NS1 gene were found to have a strong interferon (IFN) inducing phentopye. This was concluded from the finding that such transfectants were able to grow on IFN deficient Vero cells but were unable to grow on hen eggs or Madin-Darby Canine Kidney (MDCK) cells.
The influenza NS1 protein is an RNA-binding protein which has been implicated in a number of regulatory functions during influenza virus infection. It is synthesized in large amounts and found mainly in the nucleus early during infection and later in the viral cycle in the cytoplasm of the infected cells. Other than the influenza NS-1 protein, another regulatory viral protein, namely the Nef protein of HIV-1 which is a myristylated protein, is localized in the cytosol in association with the cell membrane.
An immune response directed against early expressed regulatory HIV-1 proteins could possibly allow the elimination of virus-infected host cells in the replication cycle before release of new infectious viral particles would even occur. As the Nef protein is among the first ones to be released and further is one of the major HIV proteins produced following infection, it could play a crucial role in developing an efficacious anti-AIDS vaccine.
The HIV-1 xe2x80x9cnegative factorxe2x80x9d (Nef) is encoded by an open reading frame which is located at the 3xe2x80x2 end of the virus, partially overlapping the U3 region of the 3xe2x80x2 long terminal repeat. Up to 80% of the early, multiply spliced class of viral transcripts encode Nef. The Nef gene product is an NH2-terminally myristylated protein of 27 to 30 kDa, which is predominantly localized in the cytoplasm and associated with the membrane and the cytoskeletal matrix. It is well conserved among the different human (HIV-1 and HIV-2) and Simian immunodeficiency viruses (SIV).
The close evolutionary relationship between these primate lentiviruses suggests that the Nef protein plays an important role in viral infection and pathogenesis, although the exact role in the virus life cycle and its functions at the cellular level are still the subject of current research.
Various details about the Nef protein and its effects are already known, however. For instance, it is reported that some humans infected with Nef-deleted, HIV remained disease-free, with normal CD4 counts 10 to 14 years after infection, although deletion of Nef is not a universal finding in long-term nonprogressors. In addition, Nef-deficient SIV fails to produce AIDS in infected adult macaques. SIV mutants deleted for the Nef gene even induce protection against a virulent challenge. Nef was shown to stimulate HIV-1 proviral DNA synthesis and its expression has also been found to induce the efficient internalization and degradation of the cell surface CD4 receptor for HIV-1. This Nef-induced CD4 down-regulation, which renders cells resistant to viral superinfection, has the potential to increase virus replication by facilitating release of progeny virions. It was further demonstrated that extracellular Nef protein could activate HIV-1 from latent to productive infection both in infected T-cell lines and in PBMC from asymptomatic carriers. Further, it was shown that CTLs inefficiently lysed primary cells infected with HIV-1, if the viral Nef gene product was expressed.
Protection of HIV-infected cells from efficient recognition and killing by CTLs correlates with the Nef-mediated down-regulation of MHC class I molecules. Nef also interferes with the induction of IL-2 mRNA in T-cell lines. Furthermore, there are a large number of cellular partners that have been found to be associated with Nef expression including Src family kinases, xcex2-COP, a serine-threonine kinase, thioesterase and p53.
It is also reported that the majority (about ⅔) of HIV-1 seropositive patients generated Nef-specific CTLs.
Two central multirestricted immunodominant regions (amino acids 66 to 100 and 115 to 146) and a carboxyl-terminal region (amino acids 182 to 206) were identified within the Nef protein. These three multirestricted immuno-dominant regions (amino acid sequences containing more than one T-cell epitope) are being recognized by human CD8+CTLs in association with at least 14 different MHC class I molecules including the important MHC haplotypes HLA-A1, -A3, -A11, -B8, -B17, -B18 and -B37.
The two central multirestricted domains of the Nef protein are the most highly conserved regions among different HIV-1 isolates and were imrnunodominant for most of the asymptomatic HIV-1 seropositive donors tested. In addition to the high immunogenicity of the HIV-1 Nef protein, which has been demonstrated by its capacity to induce strong T-cell immune responses, also Nef-specific B-cell immune responses are reported in the literature.
The present invention relates to the development of attenuated live vaccine vectors, more specifically to such vectors based on or derived from genetically modified influenza A virus strains. It further relates to the construction and modification of genetically engineered non-structural genes of influenza A viruses, particularly of the NS1 gene segment, wherein the modifications include deletions of selected parts of the NS1 gene segment and/or insertions of heterologous, preferably antigenic, sequences into selected sites of the NS1 gene. It is another objective of the invention to provide chimeric influenza viruses containing such modified NS1 gene segments but which do not suffer from the drawback of being IFN sensitive, in contrast to the transfectants disclosed in WO 99/64571. The invention further relates to recombinant proteins obtained from the NS1-modified viruses by expression in a suitable host system and further to a vaccine comprising the NS1-modifed viruses of the present invention.
It is yet another objective of the invention to provide a method for obtaining recombinant influenza viruses as well as attenuated influenza vaccines, based on the generation of continues cell lines (e.g. Vero, MDCK etc.) expressing synthetic influenza genes (minus sense RNA) comprising natural or engineered influenza sequences (deletions or insertions). These cell lines producing high quantities of such genes can be used for infection with influenza virus followed by selection procedures in order to get a gene of interest incorporated into the viral progeny.
The present inventors have established a reverse genetics system on Vero cells allowing them to manipulate the virulence of the PR8 influenza A virus strain by changing the length of the translated NS1 protein. In the course of the research leading to the present invention the capacity of influenza A virus to tolerate and present long insertions in the NS gene have been investigated. A collection of several chimeric NS1 gene constructs using heterologous sequences including the HIV-1 derived sequences encoding ELDKWA (SEQ ID NO: 1) of gp41 or Nef, has been established by insertion of one or more of the heterologous sequences or, optionally, several repeats of any such sequence, in frame into the NS1 protein.
The aforementioned heterologous sequences were inserted downstream nt position 400 (corresponding to aa position 124) and optionally preceded by the 2A autocleavage site sequence and/or by a leader sequence derived from the influenza HA molecule. Other constructs additionally comprised an anchor sequence derived from the influenza HA molecule as an insertion right after the desired antigenic sequence(s), thus forming the end of the entire heterologous insertion. In each case the insertions were followed by a stop codon to prevent transcription and translation of the remaining portion of the NS1 gene segment (including the effector domain), while maintaining the cleavage site for the NS splicing (necessary for transcription and translation of the NS2=NEP gene segment) fully functional.
Rescued viruses caused expression and accumulation of the foreign antigens in the cytosol and/or on the surface of the infected cells. The inventors also successfully rescued transfectant viruses harbouring a multirestricted immunodominant region rich in T-cell epitopes of HIV-1 Nef protein (136 amino acids).
All transfectants displayed normal growth characteristics in Vero cells, embryonated chicken eggs and MDCK cells, but were attenuated in mice. Chimeric influenza NS1-Nef viruses did not replicate in respiratory tracts of infected mice, but were able to induce a strong Nef-specific CTL response following a single intranasal immunization. In addition, a Nef-specific antibody response was detected following three immunizations. Transfer of the recombinant NS-nef gene by genetic reassortment from the viral PR8 (influenza A/PR/8/34; Egorov et al., 1994, Vopr. Virusol. 39:201-205) vector to other influenza strains resulted in the same level of attenuation and immunogenicity. This finding permitted the present inventors to perform effective boosting immunizations using several attenuated vectors of different antigenic subtypes.
Thus, the inventors were able to demonstrate that the approach to create a set of influenza chimeric strains belonging to different influenza subtypes while bearing the identical recombinant chimeric NS1 gene, gives the opportunity to create several strains for boosting immunizations. They have further proven that once a new chimeric NS1 gene construct is rescued it can be routinely transferred to another influenza strain by genetic reassortment.